There are two general patterns of in vitro embryogenesis: direct initiation from differentiated tissue, and indirect initiation via a callus intermediary. Direct embryogenesis proceeds from embryogenic-determined cells (Kato and Tukeuchi, 1966). Indirect embryogenesis requires dedifferentiation of embryogenic-determined cells, callus proliferation, and differentiation of embryogenic cells (Sharp et al., 1980). In vitro asexual embryogenesis occasionally occurs in the absence of exogenous hormones (Halperin and Jensen, 1967), but in most cases requires the manipulation of hormonal balance in the medium (Gamborg 1970).
Direct somatic embryogenesis from immature zygotic cacao embryos proceeds by two distinct patterns: a "budding" process in which cells of the hypocotylary epidermis develop to mimic the normal stages of embryogenesis including the development of a suspensor (Esan 1977, Pence et al. 1979, 1980) and a "nonbudding" process where a differentiation of embryos is from internal meristematic cotyledonary tissue. Direct somatic embryogenesis occurs at low frequency on basal medium alone but a combination of auxin (IAA, NAA, or 2,4-D) and coconut water stimulates the embryogenic process (Pence et al. 1979, 1980). The "budding" process continues from asexual embryos and some cultures continue to proliferate in this manner in a hormone-free medium.
Cacao callus has been initiated from various tissues including leaves (Pence et al., 1979; Searles et al., 1976), fruit (Searles et al., 1976), seedlings (Hall and Collin, 1975), and zygotic embryos (Hall and Collin, 1975; Tsai and Kinsella, 1981; Pence et al. 1979), but the only organized development from callus was limited to roots (Hall and Collin, 1975; Pence et al. 1979). This invention describes the process of asexual embryogenesis via callus tissue in Theobroma cacao L., procedures for the induction of embryogenic callus, and the histology of somatic embryogenesis. The asexual embryos so produced may then be grown for utilization as a cocoa butterlike product.